Genetic variation affecting the structure and function of the ABCA4 protein gives rise to a broad collection of genetically and clinically heterogeneous diseases, known collectively as ABCA4 disease, which are characterized by both phenotypic overlap and variable penetrance and expressivity. Some phenotypic variation can be attributed to variability of highly penetrant ABCA4 mutations; however, much of the heterogeneity cannot be explained by such mutations alone as: 1) Many patients with exactly the same ABCA4 mutations have different phenotypes, including ~67% of familial cases with temporally discordant phenotypes within a generation. 2) Patients belonging to various ethnic groups have different phenotypes even with the same mutations. 3) The pathognomonic features of ABCA4 disease vary extensively in severity, time of onset and prognostic outcome resulting in several sub-phenotypes that progress along different disease trajectories. 4) Mouse models of ABCA4 disease have variable lipofuscin accumulation phenotype depending on the genetic background. The emerging concept of Precision Medicine requires identification of all genetic variation associated with specific disease phenotypes. While the genetic analyses of ABCA4 disease have substantially advanced, an unequivocal diagnosis and accurate assessment of disease prognosis are still far from 100% efficiency. Most importantly, the genetic variation modifying the extremely heterogeneous presentation of ABCA4 disease is practically unknown. We will test two specific hypotheses: 1) Some (frequent) ABCA4 variants represent extremely hypomorphic alleles and/or modifiers, which are associated with the disease only under certain conditions. 2) Both cis- and trans-acting genetic variation beyond highly penetrant mutations modifies the expression of ABCA4 disease. The research program, based on large, comprehensively clinically and genetically characterized familial cohort of ABCA4 disease, accumulated data and knowledge to identify and functionally analyze variants in the ABCA4 locus by using a combination of human and mouse genetic analysis methods, includes two Specific Aims. In the first Aim, we increase and refine our cohort of ABCA4 disease patients with clinical and genetic methods use this and other cohorts to identify additional genetic modifiers of ABCA4 disease in the locus and across the genome. In the second Aim we will determine ABCA4 modifiers by a complementary approach utilizing the Diversity Outbred mouse mapping resource. The outcome of these studies will substantially improve the accuracy of genetic testing and prognostic assessment of the disease, and will provide a comprehensive platform for discovering therapies and blueprint for the design and structure of clinical trials.